Characterizationof soluble complexesoftheShigella£exneri type III secretion systemATPase

Shigella flexneri is the causative agent of bacillary dysentery in humans (Kotloff et al., 1999) and it uses a type III secretion system (T3SS) as its major virulence device for invasion of, and dissemination within, the gut epithelial lining (Cossart & Sansonetti, 2004). T3SSs are found in many Gram-negative bacterial pathogens, and serve as molecular injection devices to deliver bacterial virulence effector proteins into eukaryotic host target cells (Cornelis, 2006; Galan & Wolf-Watz, 2006). They are genetically, morphologically and structurally related to the basal bodies of bacterial flagella (Blocker et al., 2003). The Shigella type III secreton is encoded on a 31-kb fragment of a large virulence plasmid and consists of three major structural parts (Blocker et al., 1999): (1) a cytoplasmic region known as the ‘bulb’, (2) a region spanning both bacterial membranes termed the basal body and (3) an extracellular ‘needle’. Protein export through the T3SS requires a conserved ATPase, termed Spa47 in the Shigella T3SS and FliI in the flagellum (Dreyfus et al., 1993). In the absence of the ATPase, only inner membrane (IM) and cytoplasmic components of the T3SS are assembled (Eichelberg et al., 1994). T3SS ATPases are homologous at the sequence and structural level to the b-subunit of F1-ATP synthase, a hexameric rotary motor (Imada et al., 2007; Zarivach et al., 2007). The T3SS/flagellar ATPases have also been demonstrated to form hexamers (Claret et al., 2003; Pozidis et al., 2003; Minamino et al., 2006). It has been proposed that during the export process, the multimeric ATPase docks with other cytoplasmic components to an export complex in the IM (Zhu et al., 2002; Gonzalez-Pedrajo et al., 2006). Although the exact translocation mechanism is unknown, it has been shown that the ATPase may be involved in the release of substrates to be exported from their chaperones (Akeda & Galan, 2005). Central to the mechanism and regulation of secretion is the role of the proteins that interact with the ATPase. In flagella, it was determined, using classical genetics and recombinant protein biochemistry, that FliI interacts with two IM proteins in their cytoplasmic regions, FlhA and FlhB (MxiA and Spa40 in Shigella) (Zhu et al., 2002), and with a cytoplasmic protein, FliH, which inhibits FliI’s ATPase activity (Minamino & MacNab, 2000). FliH is homologous to MxiN in Shigella, with both displaying an evolutionary link to peripheral stalk components of the F1F0and V1V0ATPases (Pallen et al., 2006). Further work demonstrated that FliI, when overexpressed in Escherichia coli, could form